Gene-targeted mice with a simplified immune system clonal selection demands that individual B cells be monospecific. Allelic and isotypic exclusion ensure that there is but one functional heavy (H) chain and one functional light (L) chain gene. While there is general agreement that allelic and isotypic exclusion of L chain is accomplished by turning off the enzymes that assemble the genes from gene segments, various competing theories have been proposed to explain allelic exclusion at the H-chain locus. The goal of this proposal is to assess the contributions of the stochastic, genetic regulation, and cellular regulation models to the understanding of allelic exclusion at the immunoglobulin H-chain locus. Because we have obtained some indication that the pre-B-cell population contains cells with two productively rearranged H alleles, we propose to determine the frequency of the double H producers in mice with germ line H and L loci, and in the presence of preformed H and L genes. Because the products of the mu and delta alleles can be easily distinguished by immunofluorescence, we will use mice in which one C-mu allele is knocked out. Furthermore, we will test the various hypotheses in the monoclonal B-cell mouse generated by nuclear transfer from a B lymphocyte. In the proposed experiments, the H and L alleles from the original B lymphocyte will be shuffled and combined with germ line or nonfunctional alleles. The aim is to create mice that represent various pre-B- and B-cell genotypes, and to investigate the effect of the various preformed alleles on the germ line or rearranged allele(s) and on B-cell development as it relates to allelic exclusion.